SÉRIE DIGITAL

11

2016

WHY DO WE NEED HISTOLOGY OF HAKE

(Merluccius merluccius) GONADS?

THE PORTUGUESE CASE

Ana Maria Costa e Maria do Carmo Silva

RELATÓRIOS CIENTÍFICOS E TÉCNICOS DO IPMA – SÉRIE DIGITAL

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de investigação do mar e da atmosfera. Esta publicação é aberta à comunidade científica e aos

utentes, podendo os trabalhos serem escritos em Português, Francês ou Inglês.

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WHY DO WE NEED HISTOLOGY OF HAKE (MERLUCCIUS

MERLUCCIUS) GONADS? THE PORTUGUESE CASE

Ana Maria Costa e/and Maria do Carmo Silva

IPMA - Divisão de Modelação e Recursos da Pesca (DivRP)

Av. Alfredo Magalhães Ramalho, 6, 1449-006 Lisboa

Recebido em:2016.04.07 Aceite em:2016.07.12

ABSTRACT

With a monthly sampling frequency between 2010 and 2013, 4238 fish were collected and

their gonads were macroscopically and microscopically staged. The percentages of error

encountered with the macroscopic assignments were analysed by sex, length class and month,

both for all stages together and for stage 1 separately, where the highest percentages of error

are found on the identification of the immature stages that in fact were already mature. When

considering all the individuals the highest errors occurred in the females (11%), in the length

classes of 28 and 40 cm (26% and 25%, respectively) and in June (17%). The greatest

misidentifications of maturity stage 1 were verified in males in length classes under 32 cm

(92%) and over 57 cm (100%) and in the months of June, July and October (100%). The L50

decreased 8.1 cm (from 34.4 to 26.3 cm) with the microscopic identification, considering

combined sexes and 2.3 cm (from 40.6 to 38.3 cm) only in females.

Key words: Hake, histology, length-at-first maturity.

RESUMO

Título: Porque fazer histologia das gónadas de pescada (Merluccius merluccius)? O caso português Com uma amostragem mensal entre 2010 e 2013 foram recolhidos 4238 exemplares e foi

atribuído o seu estado de maturação macroscópico e microscópico. As percentagens de erro

encontradas na identificação macroscópica foram analisadas por sexo, classe de comprimento

e mês, para todos os estados de maturação em conjunto e para o estado 1 em separado, tendo

as maiores percentagens de erro sido encontradas na identificação dos estados imaturos em

gónadas que na realidade já estavam maturas. Considerando todos os indivíduos, os maiores

erros ocorreram nas fêmeas (11%), nas classes de comprimento dos 28 e 40 cm (26% e 25%,

respectivamente) e em Junho (17%). O maior erro na atribuição do estado 1 verificou-se nos

machos (92%), nas classes de comprimento abaixo dos 32 cm (92%) e acima dos 57 cm

(100%) e nos meses de Junho, Julho e Outubro (100%). O L50 decresceu 8.1 cm com a

identificação microscópica, de 34,4 para 26,3 cm, considerando os sexos combinados e 2,3

cm nas fêmeas, de 40,6 para 38,3 cm.

Palavras-chave: Pescada, histologia, comprimento de primeira maturação.

REFERÊNCIA BIBLIOGRÁFICA COSTA, A.M. and SILVA, M.C., 2016. Why do we need histology of hake (Merluccius merluccius)

gonads? The Portuguese case. Relat. Cient. Téc. do IPMA (http://ipma.pt) nº 11 14 p.

3

INTRODUCTION

Knowledge of the dimension of the reproductive stock is essential for the assessment of the

state of exploitation of fish resources (Bromley, 2003). This evaluation is based on the

combined modelling of biological information (reproduction, growth, feeding habits, among

others) and fisheries (fishing effort, catches, landings and discards) (Rothschild et al., 1987;

Sparre et al., 1989; Shepherd, 1993). However, the biological characteristics must be the

fundamental basis for the conservation and management of marine living resources (Cadima,

2000). In any reproductive study the first step is the identification of the sex of the individual,

and the identification of the level of maturation is the subsequent one (Vazzoler, 1996). West

(1990) points out that the identification of the reproductive cycle of a species is made by

classifying the development of the gonads and Guerra and Sánchez (1998) state that the study

of the maturation of the gonads is an essential item for the identification of reproductive seasons

and areas, of the achievement of the gonads’ first maturation and of fecundity. One of the main

criteria for assessing the state of exploitation of a fishery relies on the knowledge of spawning

stock biomass (SSB) (Bromley, 2003), forwhich the maturation stage is its principal

component. To know the spawning season and the number of mature and immature fish of a

certain species, information about the maturity stages of those individuals is necessary. To

obtain the maturity ogives it is necessary to correctly identify maturity stages which present

specific characteristics of oocyte developmental status in the case of the female gonads and

spermatozoa in the male gonads. The correct assignment of the maturity stage is extremely

important since errors in its determination may lead to an incorrect estimation of the spawning

biomass and therefore of the exploitation status of the stock. Gonad maturity stages are

usually assigned by macroscopic observation of their external characteristics. This

methodology allows the analysis of a great number of individuals with a reduced effort and in

a very short time period. However, doubts raised by some experienced observers justified a

review of the maturity scale by contrast to histological observation. In particular, doubts were

raised regarding the occurrence of immature individuals much larger than expected for first

maturity, which therefore may be resting, and externally present gonads of characteristically

immature individuals (Morgado and Gonçalves, 2007). These issues led to the development of

a microscopic maturity scale formerly with five stages (Gonçalves et al., 2004) and finally

converted to four stages (Morgado and Gonçalves, 2007), which must always be applied in

order to correctly distinguish between mature and immature individuals.

The aim of this paper is to demonstrate the need of histology to assign maturity stages to hake

gonads, and the implications of its application in the correct definition of the spawning season

4

and in the determination of length at first maturity.

MATERIAL AND METHODS

This study is based on the observation of 4238 individuals (1233 males, 2870 females and 135

indeterminate specimens) caught from 2010 to 2013 by the fishing fleets that land on the

Portuguese coast in Póvoa do Varzim, Matosinhos, Peniche and Olhão. The characteristics of

the samples studied each month are indicated in Table 1. All fish were measured (total length

in cm), weighted (total weight in g), sexed and the maturity stage was assigned according with

the four stage maturity scale (ICES, 2007). Gonads were removed and fixed in 10%

formaldehyde neutralized with phosphate salts for subsequent histological processing.

Sections of 3-5 µm were stained with hematoxylin-eosin and analysed under a light

microscope to determine microscopic maturity stage according to Morgado and Gonçalves

(2007). The macroscopic and microscopic characteristics of each maturity stage are given in

Table 2. The error in the macroscopic identification was evaluated by sex, length class and

month, not only taking into account all fish sampled but also considering only macroscopic

stage 1, which correct identification is crucial to get the real percentage of mature and

immature specimens. Due to the importance of knowing the error in the length at first

maturity induced by incorrect determination of maturity, macroscopic and microscopic

maturity ogives were designed considering only the females and both sexes combined (the

current method used in hake assessment).

Table 1 - Number of fish sampled per month

Tabela 1 - Número de peixes amostrados por mês

YearMonth

TotalLength

range (cm)Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

2010 51 98 119 178 131 24 52 128 178 41 239 1239 20.7-76.7

2011 191 49 89 120 187 52 103 38 93 5 163 44 1134 25.0-76.5

2012 167 101 62 36 76 41 95 34 69 80 119 70 950 23.0-92.0

2013 172 91 75 32 179 15 46 57 23 50 60 115 915 23.0-86.1

5

Table 2 - Macroscopic and microscopic characteristics of each maturity stage

Tabela 2 - Características macroscópicas e microscópicas de cada estado de maturação

Mat

urity

stag

eM

acro

scop

ic a

ppea

ranc

e - F

emal

esM

icro

scop

ic a

ppea

ranc

e - F

emal

es

1. I

mm

atur

e / R

estin

gS

mal

l o

var

ies,

wit

h fi

rm c

onsi

sten

ce a

nd

min

imal

vas

cula

riza

tio

n, t

ransp

aren

t o

r p

inkg

rey,

wit

ho

ut

op

aqu

e

or

hya

lin

e o

ocy

tes

Th

is s

tag

e in

clud

es t

wo

sta

ges

that

are

no

t m

acro

sco

pic

ally

dis

tingu

ish

able

-

imm

atu

re a

nd r

esti

ng

. In

th

e im

mat

ure

fem

ales

all

the

oo

cyte

s in

the

ov

ary

are

in p

rim

ary

gro

wth

sta

ge

and

the

oo

cyte

s ar

e w

ell p

ackag

ed.

The

rest

ing

ovar

y d

oes

n't

pre

sent

mat

ure

oo

cyte

s, h

as a

wid

e o

var

y w

all,

lam

ella

e ar

e

no

t as

co

mp

act

as i

n im

mat

ure

ov

arie

s an

d b

loo

d v

esse

ls m

ay b

e m

ore

abund

ant

2. D

evel

opin

g /

Mat

urin

gM

ediu

m o

r la

rge o

var

ies,

pin

k o

r yel

low

to

ora

nge,

wit

h

vas

cula

riza

tio

n v

aria

ble

, pre

sen

t an

d o

bvio

us.

Op

aque

oo

cyte

s p

rese

nt b

ut

wit

ho

ut

hyal

ine

oo

cyte

s

Th

is s

tag

e is

char

acte

rize

d b

y th

e o

ccurr

ence

of

cort

ical

alv

eoli

and

/or

vit

ello

gen

ic o

ocy

tes,

but

PO

Fs

are

no

t p

rese

nt

and

no s

igns

of

advan

ced

spaw

nin

g p

roce

sses

, su

ch a

s a

thic

k o

var

y w

all,

hig

h vas

cula

riza

tio

n o

f

go

nad

and

/or

dis

org

aniz

atio

n o

f la

mel

lae

3. S

paw

ning

A -

Hyd

rate

d - L

arg

e o

var

ies,

wit

h fi

rm c

onsi

sten

ce a

nd

vas

cula

riza

tio

n, p

ink

or

red

ish

ora

nge.

Op

aqu

e an

d

hya

line

oo

cyte

s p

rese

nt

Pre

sence

of

hig

h p

erce

nta

ge

of

hyd

rate

d o

ocy

tes

or

at t

he

beg

innin

g o

f th

e

hyd

rati

on

pro

cess

B -

Part

ial s

paw

ning

- L

arge

ovar

ies,

fla

ccid

, w

ith

vas

cula

riza

tio

n, p

ink

or

red

ish

ora

nge.

Wit

h o

paq

ue

oo

cyte

s p

rese

nt b

ut

wit

ho

ut

hyal

ine

oo

cyte

s

PO

Fs

are

ob

serv

ed t

hro

ug

ho

ut

the

ov

ary

tog

ether

wit

h v

itel

logen

ic o

ocy

tes

in d

iffe

rent st

ages

. T

her

e ar

e no s

ign

s o

f ad

van

ced

sp

awnin

g p

roce

ss,

such

as h

igh

num

ber

of

blo

od

ves

sels

, sw

elli

ng

ov

ary

wal

l, a

tres

ia,

dis

org

aniz

atio

n o

f o

var

y s

truct

ure

s, e

tc.

4. P

ost-

spaw

ning

Sm

all

or

med

ium

ovar

ies,

fla

ccid

, d

ark

pin

k, o

rang

e o

r

purp

le.

Op

aqu

e an

d h

yali

ne

oo

cyte

s ab

sent

or

resi

dual

Fem

ales

at

this

sta

ge

wil

l no l

onger

pro

duce

mo

re o

ocy

tes

to b

e re

leas

ed

duri

ng

the

curr

ent

bre

edin

g se

aso

n.

They

are

ch

arac

teri

zed

by

a hig

h le

vel

of

atre

sia,

old

PO

Fs,

dis

org

aniz

atio

n o

f o

var

y s

truct

ure

s, n

um

ero

us

blo

od

ves

sels

, th

ick

ov

ary

wal

l an

d a

bse

nce

of

yo

lked

oo

cyte

s gro

up

s (e

xce

pti

ng

som

e at

reti

c yo

lked

oo

cyte

s)

Mat

urity

stag

eM

acro

scop

ic a

ppea

ranc

e - M

ales

Mic

rosc

opic

app

eara

nce

- Mal

es

1. I

mm

atur

e / R

estin

gS

mal

l te

stis

, tr

ansp

aren

t o

r w

hit

e, w

ith t

he

shap

e o

f a

thin

rib

bo

n, w

ith

no s

igns

of

dev

elo

pm

ent.

Wit

ho

ut s

per

m

Th

ere

are

only

sp

erm

ato

go

nia

and

pri

mar

y sp

erm

ato

cyte

s p

rese

nt

2. D

evel

opin

g /

Mat

urin

gM

ediu

m t

esti

s, w

ith t

he

shap

e o

f d

evel

op

ban

ds.

Sper

m f

low

s

when

tes

tis

are

cut

Th

e p

rese

nce

of

seco

nd

ary

sper

mat

ocy

tes

and

sp

erm

atid

s is

th

e m

ain

char

acte

rist

ic

3. S

paw

ning

Lar

ge

whit

e te

stis

, w

ith t

he

shap

e o

f la

rge

ban

ds.

Sp

erm

flo

ws

wit

h p

ress

ure

on

the

abd

om

en

Sem

inif

ero

us

tub

ule

s ar

e th

ick

and

full

of

sper

mat

ozo

a

4. P

ost-

spaw

ning

Lar

ge

test

is,

whit

e o

r li

gh

t p

ink

, em

pty

and

def

orm

ed.

Sp

erm

ab

sent

or

resi

dual

Sem

inif

ero

us

tub

ule

s ar

e em

pty

, w

ith

som

e re

sid

ual

s sp

erm

ato

zoa.

Sp

erm

ato

go

nia

are

pre

sent in

the

test

is c

ort

ex

6

RESULTS

The results of the comparison between macroscopic and microscopic assignment highlight

differences in the macroscopic identification of immature fish which have been found to be

mature when analysed microscopically. The highest error occurred in females, 11%, against

6% in males, which results in a 10% error when considering all individuals sampled (Figure

1).

Figure 1 - Percentage of error of macroscopic identification by sex (all stages considered)

Figura 1 - Percentagem de erro na identificação macroscópica por sexo (considerados todos os

estados)

Since, as explained above, the macroscopic distinction of mature and immature stage 1 is

difficult, the analysis was repeated taking into account only the macroscopic stage 1

assignements, which lead to different results, as shown in Figure 2. In this case the percentage

of error was extremely high 84% with sexes combined, 82% for females and 92% for males.

Figure 2 - Percentage of error of macroscopic identification by sex (stage 1)

Figura 2 - Percentagem de erro na identificação macroscópica por sexo (estado 1)

7

The analysis by length class show highest percentage error between 26 and 45 cm, with a

maximum of 26% and 25% in the 28 and 40 cm length classes, respectively. Individuals under

26 cm and over 63 cm were all correctly identified (Figure 3).

Figure 3 - Percentage error in macroscopic identification by length class (all stages)

Figura 3 - Percentagem de erro na identificação macroscópica por classe de comprimento

(todos os estados)

Considering once again only macroscopic stage 1, the distribution of errors is very different

(Figure 4): all fish bellow 32 cm and above 57 cm were identified as immature but in fact they

were all mature. On the other hand, 47, 53 and 54 cm length classes contained no mature

individuals although identified as such macroscopically.

Figure 4 - Percentage of error in macroscopic identification by length class (stage 1) Figura 4 - Percentagem de erro na identificação macroscópica por classe de comprimento (estado 1)

8

About the monthly distribution of all maturity stages there is a great similarity between

macroscopic and microscopic identifications (Figure 5), although the latter showed smaller

percentages of immature fish in the samples.

Figure 5 - Percentages of mature and imature individuals (macroscopic (a) and microscopic

(b) identification)

Figura 5 - Percentagem de indivíduos maturos e imaturos (identificação macroscópica (a) e

microscópica (b))

To understand the error introduced in these identifications the analysis of Figure 6 shows that

in both cases the highest errors occurred out of the spawning season (17% in June and 14% in

July for all stages), reaching 100% in June, July and October when considering only

macroscopic stage 1.

Figure 6 - Percentage of error of macroscopic identification by month of all stages

(left) and of stage 1 alone (right)

Figura 6 - Percentagem de erro na identificação macroscópica por mês de todos os estados

(esquerda) e apenas do estado 1 (direita)

The analysis of the distribution of the maturity stages by month (Figure 7) shows that there is

no great discrepancy between macroscopic and microscopic identifications for males. On the

9

other hand, regarding the females the microscopic identification reveals some data not

displayed with the macroscopic staging: the macroscopic maturity stage 1 (immature) actually

includes not only immature individuals but also resting ones (mature); in August and

September there were fish spawning (Stage 3A), which were not identified macroscopically

and the post-spawning fish (Stage 3B) occurred all year round and not only during the

spawning season; the high percentages of resting fish (Stage 4) which were identified just

prior to the spawning season were not found by microscopic analysis.

Figure 7 - Monthly distribution of macroscopic and microscopic maturity stages

Figura 7 - Distribuição mensal dos estados de maturação macroscópicos e microscópicos

10

Length at first maturity also showed differences, not only between determinations made from

all individuals (males and females) versus only females, but also between the ones obtained

under, macroscopic and microscopic observations, as shown in Figure 8 and Table 3.

Figure 8 - Macroscopic and microscopic maturity ogives of combined sexes (N = 4103) and

only females (N = 2870)

Figura 8 - Ogivas de maturação macroscópicas e microscópicas de sexos combinados (N =

4103) e só fêmeas (N = 2870)

Table 3 - Parameters of the maturity ogives of combined sexes and only females

Tabela 3 - Parâmetros das ogivas de maturação de sexos combinados e só de fêmeas

The correct identification of mature and immature fish through the microscopic observation

leads to an L50 lower than the one obtained under macroscopic identification - 8 cm for sexes

combined and 2 cm for females only.

Macroscopic id. Microscopic

Sexes combined

0 10 20 30 40 50 60 70 80 90

Length class (cm)

0.00

0.25

0.50

0.75

1.00

1.25

Pro

port

ion m

atu

re

Females

0 10 20 30 40 50 60 70 80 90

Length class (cm )

0.00

0.25

0.50

0.75

1.00

1.25

Pro

po

rtio

n m

atu

re

Macroscopic identification Microscopic identification

Sexes combined Females Sexes combined Females

a 4.5156 11.1032 2.3670 9.6926

b 0.131 0.273 0.090 0.253

R 0.952 0.994 0.921 0.994

L50 34.4 40.6 26.3 38.3

11

DISCUSSION

The maturity scales proposed by Lucio et al. (2000) and Gonçalves et al. (2004) do not

separate immature from resting individuals since they are not distinguishable macroscopically,

which may lead to an underestimation of the spawning biomass. Adult hake which at the end

of the spawning season are resting (macroscopically similar to the immature stage), will not

be considered in the determination of SSB, although they have contributed to the reproductive

potential of that year. Similarly to other species, such as Gadus morhua (Vitale et al., 2006),

this is a common issue also to other species of the genus Merluccius, for which the use of

histology is essential for the correct identification of the immature and resting maturity stages,

as referred by Honji et al. (2006), Louge and Christiansen (1993), Louge (1996) and

Vaz-dos-Santos et al. (2005) for the argentine hake (Merluccius hubbsi). The application of

histology to the gonads of adult individuals of different geographic areas and periods

combined with the distribution of eggs and larvae of the species can at the same time provide

some important information about the migratory behaviour of that species (Christiansen et al.,

1986). At the same time, histology also allows a verification of the synchrony or asynchrony

of ovarian development, which is valuable information to characterise a multispawner species,

as reported by Balboutin and Fischer (1981) and Goldberg (1985) for Merluccius gayi gayi.

Regarding stock management, the error introduced by the incorrect assignment of the mature

and immature stages may have a strong impact on stock size progression estimates.

This work shows that the monthly distribution of maturation states and consequent

identification of spawning season is not much different considering macroscopic and

microscopic identifications. The value obtained macroscopically for the length at first

maturity of females, 40.6 cm, is slightly lower than the ones indicated in other references

(41.1 cm in Martos et al., 1996, for the Northwest African waters; 44.1 cm in Cardador,

unpublished, for the Portuguese waters; 45.4 cm in Piñeiro and Saínza, 2003, for the

Northwest Iberian Peninsula; 47.1 cm (in 2003) and 45.7 cm (in 2004) in Domínguez-Petit,

2006, on the Galician shelf; 46.5 cm in Domínguez-petit et al., 2008, for Galicia and the Bay

of Biscay; 32.5 cm in Al-Absawy, 2010, for Egyptian Mediterranean waters; 45-50 cm in

Murua, 2010, for Galicia and the Bay of Biscay; 41.3 cm in Costa, 2014, for the Portuguese

coast and 21.5 cm in Soykan et al., 2015, for the Central Aegean Sea, Turkey), as well as the

34.4 cm for sexes combined (36.0 cm in Cardador, unpublished; 37.9 cm in Piñeiro and

Saínza, 2003 and 34.5 cm in Costa, 2014). However, the microscopic analysis corrects these

figures allowing a more accurate assessment of the Iberian hake stock.

So, the results obtained with this study seem to indicate that in order to get a good assessment

12

of the stock of hake all states considered immature macroscopically should be examined

histologically.

ACKNOWLEDGEMENTS

This work was supported by the PNAB/EU DCR-Data Collection Regulation. The authors are

grateful to the technicians that along the years have performed the biological sampling that

made this work possible and in particular to Paula Abreu, Mónica Inácio and Daniel Pinto for

the histological preparation of the samples.

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